As pivotal regulators of apoptotic cell death, the logic of manipulating BCL-2 functions for anti-tumor effects is perhaps the strongest for any of the molecular targets proposed for cancer therapeutics. BCL-2 survival proteins, such as BCL-2 and BCL-XL are highly expressed in many types of tumors compared to normal tissue, and confer resistance to many different cytotoxic stimuli. We have identified a set of small molecules based on Antimycin A (AA), which selectively induce rapid cytotoxic response in cells expressing high levels of BCL-XL or BCL-2, suggesting a gain of function due to this interaction. The objective of this application is to develop methods to enrich for compounds with AA-like activity against cells expressing high levels of BCL-2 survival proteins. The first screen is to test compounds for cytotoxicity at a single concentration against a cell line that is engineered to overexpress BCL-XL. This will be the initial identification of hits, and will be compared to the activity of a 2-methoxy derivative of AA, on those same cells. The secondary screen will be testing the hits in a side-by-side comparison of isogenic cell lines expressing high and low levels of BCL-XL. Comparison of IC50 values will determine the selectivity of the hit compounds for killing high BCL-XL expressing cells. The counter-screen will involve a competitive binding assay based on the anisotropy changes measured when AA binds to BCL-XL. This is to determine which of the hits from the first two screens bind to the same binding site as AA, and will also serve to determine the relative affinity of that interaction. This progressive strategy will identify compounds that induce a selectively cytotoxic response in cells expressing high levels of BCL-XL, compared to cells expressing low levels of BCL-XL, and determine which of these compounds works by interacting with the same binding site as AA. Since AA derivatives are the only compounds identified to date that selectively kill cells expressing high levels of BCL-XL compared to normal endogenous levels, the identification other compounds that work similarly, is crucial to exploiting this as a new class of therapeutics. The ultimate aim of this is the development of novel therapeutics for the treatment of drug-resistant tumors.